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University Department of Surgery, Blond McIndoe Centre, Royal Free and University College Medical School, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UKDepartment of Surgical and Perioperative Science, Section for Hand and Plastic Surgery, Umeå University Hospital, Umeå, SwedenDepartment of Integrative Medical Biology, Section for Anatomy, Umeå University Hospital, Umeå, Sweden
University Department of Surgery, Blond McIndoe Centre, Royal Free and University College Medical School, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UKDepartment of Surgical and Perioperative Science, Section for Hand and Plastic Surgery, Umeå University Hospital, Umeå, SwedenDepartment of Integrative Medical Biology, Section for Anatomy, Umeå University Hospital, Umeå, Sweden
Department of Surgical and Perioperative Science, Section for Hand and Plastic Surgery, Umeå University Hospital, Umeå, SwedenDepartment of Integrative Medical Biology, Section for Anatomy, Umeå University Hospital, Umeå, Sweden
Corresponding author. Address: Blond McIndoe Research Laboratory, Plastic and Reconstructive Surgery Research, University of Manchester, Room 3.102, Stopford Building, Oxford Road, Manchester M13 9PT, UK. Tel.: +44-161-2751594; fax: +44-161-275-1591
University Department of Surgery, Blond McIndoe Centre, Royal Free and University College Medical School, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK
The loss of a large proportion of primary sensory neurons after peripheral nerve axotomy is well documented. As a consequence of this loss, the innervation density attained on completion of regeneration will never be normal, regardless of how well the individual surviving neurons regenerate. Acetyl-l-carnitine (ALCAR), an endogenous peptide in man, has been demonstrated to protect sensory neurons, thereby avoiding loss after peripheral nerve injury. In this study we examined the dose-response effect of ALCAR on the primary sensory neurons in the rat dorsal root ganglia (DRG) 2 weeks after sciatic nerve axotomy.
Six groups of adult rats (n=5) underwent unilateral sciatic nerve axotomy, without repair, followed by 2 weeks systemic treatment with one of five doses of ALCAR (range 0.5–50 mg/kg/day), or normal saline. L4 and L5 dorsal root ganglia were then harvested bilaterally and sensory neuronal cell counts obtained using the optical disector technique. ALCAR eliminated neuronal loss at higher doses (50 and 10 mg/kg/day), while lower doses did result in loss (12% at 5 mg/kg/day, p<0.05; 19% at 1 mg/kg/day, p<0.001; 23% at 0.5 mg/kg/day, p<0.001) compared to contralateral control ganglia. Treatment with normal saline resulted in a 25% (p<0.001) loss, demonstrating no protective effect in accordance with previous studies.
ALCAR preserves the sensory neuronal cell population after axotomy in a dose-responsive manner and as such, has potential for improving the clinical outcome following peripheral nerve trauma when doses in excess of 10 mg/kg/day are employed.
but application in the clinical setting has not been feasible. Acetyl-l-carnitine (ALCAR), the acetyl ester of l-carnitine and predominant acyl-carnitine in human tissues, is neuroprotective in vitro
Systemic acetyl-l-carnitine eliminates sensory neuronal loss after peripheral axotomy: a new clinical approach in the management of peripheral nerve trauma.
Along with carnitine, ALCAR plays a role in facilitating the transport of long-chain free fatty acid transport molecules across inner mitochondrial membranes.
Immunohistochemical quantification of cutaneous innervation in HIV-associated peripheral neuropathy: a study of acetyl-l-carnitine therapy. Abstract 36. Third International Workshop on Salvage for HIV Infection, Chicago 2000.
Immunohistochemical quantification of cutaneous innervation in HIV-associated peripheral neuropathy: a study of acetyl-l-carnitine therapy. Abstract 36. Third International Workshop on Salvage for HIV Infection, Chicago 2000.
Systemic acetyl-l-carnitine eliminates sensory neuronal loss after peripheral axotomy: a new clinical approach in the management of peripheral nerve trauma.
However, the profile of neuron death at doses below this, which may still be beneficial while reducing the cost of potential clinical use of ALCAR, has yet to be ascertained. The purpose of our present study was therefore to investigate the correlation between the dose of ALCAR and the extent of neuronal rescue, as quantified by the validated
All work was performed in accordance with the terms of the Animal (Scientific Procedures) Act 1986 and the experimental design minimised the number of experimental animals.
1.1 Animal model and experimental design
Under halothane anaesthesia (May and Baker Ltd., UK) (2 ml min−1 in oxygen), six groups of young adult Sprague-Dawley rats (250–300 g body weight) underwent unilateral sciatic nerve division at the upper border of quadratus femoris (n=5 per group). To prevent spontaneous regeneration, both proximal and distal nerve stumps were ligated and secured into blind-ending silicon caps with 9/0 Ethilon sutures.
Each group was then randomly assigned to receive differing doses of parenteral systemic therapy with ALCAR (SigmaTau Pharmaceuticals, Italy) dissolved in normal saline, administered in the form of a 1 ml intraperitoneal injection immediately postoperatively and once daily thereafter during a survival period of 2 weeks. The doses administered were: 50, 10, 5, 1 and 0.5 mg/kg. A sixth ‘sham treatment’ group received a daily 1 ml intraperitoneal injection of normal saline.
A further three groups were operated upon in the same manner as described above for electron microscopic studies. ALCAR was administered intraperitoneally at doses of 50 or 1 mg/kg/day to two of these groups. The third, ‘sham treatment’ group, received normal saline.
1.2 Tissue processing for neuron counts
Harvesting of the ipsilateral axotomised and contralateral control L4 and L5 dorsal root ganglia (DRG) was performed under terminal anaesthesia (pentobarbitone 240 mg/kg i.p.), after transcardial perfusion with 300 ml of phosphate buffered saline (PBS) followed by perfusion fixation with 400 ml of 4% (w/v) paraformaldehyde. The DRGs were then post-fixed in 4% paraformaldehyde at 4 °C overnight before equilibration in cryoprotectant solution of PBS containing 15% sucrose and 0.1% sodium azide.
The ganglia were subsequently blocked into O.C.T. compound (BDH Laboratory Supplies) and stored at −40 °C in preparation for sectioning. Each ganglion was cut serially into 30 μm cryosections, and after permeabilisation with 0.2% TritonX-100, the sections were stained with the non-specific nuclear stain Hoechst 33342 (H33342, Sigma, Poole, UK), and with propidium iodide (PI, Sigma, Poole, UK) which highlights cytoplasmic morphology.
For electron microscopy (EM), DRGs were harvested using the same protocol, except that only 50 ml of PBS was used and perfusion fixation was achieved with 500 ml of 3% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer. The L4+L5 DRGs were post-fixed in this same solution at 4 °C, then immersed in 1% osmium tetroxide at 37 °C for 45 min, dehydrated in acetone and embedded in Vestopal. The blocks were trimmed on a Pyramitome (LKB, Sweden) and sectioned with a 2128 Ultratome (LKB). The ultrathin (60–70 nm) sections were cut and collected on formvar-coated one-hole copper grids, stained with uranyl acetate and lead citrate. Neurons were examined, and representative photomicrographs obtained using a JEOL 100CX electron microscope. Using an AGFA scanner, films were digitised to 1200 dpi output resolution, resized, grouped into a single canvas and labelled using Adobe Photoshop 7.0 software. Contrast and brightness were then adjusted to provide optimal clarity.
was used in conjunction with a CAST (computer assisted stereology toolbox)-Grid (version 1) system and an Olympus BX50 microscope. The number of neurons in each ganglion were counted, and the number of surviving L4+L5 DRG neurons estimated using previously published protocols.
Systemic acetyl-l-carnitine eliminates sensory neuronal loss after peripheral axotomy: a new clinical approach in the management of peripheral nerve trauma.
In essence the combined L4+L5 neuron counts were recorded for both axotomised and contralateral control sides, and neuron loss calculated by subtracting the neuron count in axotomised L4+L5 ganglia from that of their contralateral L4+L5 controls. Mean loss was then calculated for each dose of ALCAR, and expressed as a percentage of the mean neuron count in contralateral control ganglia.
1.4 Neuron-containing DRG volume
The neuron-containing volume of each DRG was also calculated in accordance with previous protocols.
Volume loss on the axotomised side was calculated by subtracting the average combined L4+L5 volumes from the corresponding figure for contralateral, non-axotomised, control L4+L5 DRGs and was expressed as a percentage of the value for the control side.
1.5 Statistics
Sigmastat 2.0 software package was used to analyse the results. Comparison between groups was performed using ANOVA and pair-wise comparisons were made using Student's t-test or the Mann–Whitney rank sum test when data were not normally distributed.
2. Results
No adverse reactions were observed, nor did any change in animal behaviour occur as a result of ALCAR treatment.
2.1 Neuronal loss
The effect of the varying doses of systemic ALCAR treatment upon the number of neurons within L4 plus L5 DRGs after unilateral sciatic nerve axotomy is summarised in Fig. 1. The mean neuron counts (L4 plus L5) in the non-axotomised contralateral control ganglia in the sham group at 2 weeks postoperatively was 32,199 (SD 400). This correlated well (P=0.071 for ANOVA multiple group comparisons) with the equivalent contralateral ganglia in the 50 mg/kg ALCAR group (31,401, SD 1292), the 10 mg/kg group (30,524, SD 1619), the 5 mg/kg group (30,380, SD 1591), the 1 mg/kg group (29,961, SD 1174) and the 0.5 mg/kg group (31,351, SD 554).
Fig. 1Dose-related effect of ALCAR on neuron loss 2 weeks post axotomy. N=5/group. Axotomised and contralateral control ganglia neuron counts are plotted as mean (and SD) count per experimental group. Y-axis begins at a mean neuronal cell count of 20,000. *P=<0.05, **P<0.001 axotomised vs. contralateral non-axotomised control within each group. For 50 mg/kg/day group P=0.596. For 10 mg/kg/day group P=0.559.
Neuron loss in axotomised ipsilateral L4 plus L5 ganglia in the sham group was 25% (mean counts 24,053, SD 1497, P<0.001 compared to contralateral). Neuronal loss in the ipsilateral DRG in the 0.5 mg/kg group was 23% (mean count 23,997, SD 1247, P<0.001), and was 19% (mean count 24,138, SD 1106, P<0.001) in the 1 mg/kg group. A significant loss of 12% still occurred in the 5 mg/kg group (mean count 26,583, SD 2017, P<0.05), but preservation of the neuron population was achieved in both the 10 and 50 mg/kg groups. A small 2% loss occurred in the 10 mg/kg group (mean 30,063, SD 931) but these counts did not differ significantly from the contralateral non-axotomised control (P=0.559). A minimal gain of 2% occurred in the 50 mg/kg group (mean 31,937, SD 1479), but again this value did not differ from contralateral control (P=0.596).
2.2 DRG volumes
The changes in volume of the neuron-containing region of the ganglia that occurred as a result of peripheral axotomy for the differing doses of ALCAR are shown in Fig. 2. Axotomy caused a reduction in L4+L5 DRG volume, the extent of which varied with ALCAR dose, with results reflecting the magnitude of neuronal loss (Fig. 1). A dose response was evident in volume loss at all doses, although loss only reached statistical significance at 1 mg/kg/day or less (50 mg/kg/day 0.07% gain, P=0.986 vs. contralateral ganglia; 10 mg/kg/day 2.6% loss, P=0.561; 5 mg/kg/day 9.9% loss, P=0.111; 1 mg/kg/day 12.2% loss, P=0.012; 0.5 mg/kg/day 23.8% loss, P<0.001. Volume loss was 26.3% (P=0.021 vs. contralateral ganglia) in the sham treated group.
Fig. 2Dose-related effect of ALCAR on neuron-containing DRG volume 2 weeks post axotomy. N=5/group. The volume loss in the axotomised ganglia is shown as the percentage of the mean volume in the contralateral control side. *P<0.05, **P<0.001 axotomised vs. non-axotomised contralateral control within each group.
When compared to normal neurons with intact axons (Fig. 3(A)) , the majority of neurons within sham treated ganglia exhibit grossly disrupted cytoplasmic architecture, particularly with regard to mitochondrial and endoplasmic reticular (ER) morphology (Fig. 3(B)). Such changes range from mitochondrial swelling and partial dissolution of the ER, which may be part of the regenerative, chromatolytic response, to features more compatible with cell death. These include increased electron density of the cytoplasm, and loss of mitochondrial architecture that ranged from cristal disruption, to vacuolation or actual rupture. The morphology of cellular organelles was improved by all doses of ALCAR studied, and although some degree of mitochondrial cristal disruption occurred with 1 mg/kg/day, morphology was significantly more normal with 50 mg/kg/day, a degree of mitochondrial swelling and ER dissolution being the only consistent abnormalities.
Fig. 3Electron photomicrographs of dorsal root ganglion (DRG) neurons. An intact, non-axotomised neuron is shown in panel A, whose normal morphological features include a well-defined rough endoplasmic reticulum (short black arrow), and healthy mitochondria, with intact cristae. Representative photomicrographs of axotomised neurons are also shown two week after sciatic nerve transection, during which time daily treatment with saline (B) and or ALCAR 1 mg/kg/day (C) or 50 mg/kg/day (D) ALCAR was administrated. Saline treated neurons exhibit increased electron density in cytoplasm (white transparent arrow), disrupted mitochondrial architecture (white opaque arrow) and partial disappearance of rough endoplasmatic reticulum. ALCAR causes a dose-dependent improvement in the cells' ultrastructure with progressive improvements in rough endoplasmic reticulum and mitochondrial morphology. Some mitochondria remain swollen (white dotted arrow) in both ALCAR treated groups, while unlike 50 mg/kg/day, a dose of 1 mg/kg/day treatment fails to preserve cristal architecture in the majority of mitochondria (long black arrow). Scale bar equals 1 μm.
The results of this study clearly indicate that a dose-response relationship exists between systemic ALCAR administration and the prevention of neuron cell loss after peripheral nerve division.
Capping and burial of both the proximal and distal nerve stumps after nerve division at the mid-thigh level was undertaken as a measure to prevent spontaneous regeneration and to remove any influence of distal stump-derived neurotrophic factors, thereby isolating more definitively the DRG neurons from any other neuroprotective effects.
such as age and proximity of nerve division to the ganglion.
In previous experiments 2 weeks after nerve division was identified as the time when the rate of neuronal death is maximal and when neuron loss first becomes very highly significant if the nerve is left unrepaired.
Consequently, by employing the 2 week time point, any protective effect of ALCAR, even at lower doses, should be clearly identifiable. The technique of optical dissection has been validated against serial reconstruction counts of DRG neurons
The upper limit of the ALCAR dose range was set at 50 mg/kg/day as this dose has been shown to prevent neuronal death and eliminate loss in a similar model,
Systemic acetyl-l-carnitine eliminates sensory neuronal loss after peripheral axotomy: a new clinical approach in the management of peripheral nerve trauma.
Acetyl-l-carnitine deficiency as a cause of altered nerve myo-inositol content, Na, K-ATPase activity, and motor conduction velocity in the streptozotocin-diabetic rat.
Although both 50 and 10 mg/kg/day prevented neuron loss in this model, we suggest 50 mg/kg/day as the optimum ALCAR dose since limited neuron death, which will eventually lead to neuron loss, was previously identified with the administration of 10 mg/kg/day.
Systemic acetyl-l-carnitine eliminates sensory neuronal loss after peripheral axotomy: a new clinical approach in the management of peripheral nerve trauma.
Systemic acetyl-l-carnitine eliminates sensory neuronal loss after peripheral axotomy: a new clinical approach in the management of peripheral nerve trauma.
Immunohistochemical quantification of cutaneous innervation in HIV-associated peripheral neuropathy: a study of acetyl-l-carnitine therapy. Abstract 36. Third International Workshop on Salvage for HIV Infection, Chicago 2000.
As the dose was steadily reduced, there was a mirrored reduction in the protective effect of ALCAR. Statistically significant neuron loss occurred at a dose of 5 mg/kg/day with increasingly significant loss at doses below this. The neuroprotective effect was due to ALCAR, and not to the process of pharmacotherapy administration, since neuron loss in the sham group is equivalent to that found after no treatment.
Systemic acetyl-l-carnitine eliminates sensory neuronal loss after peripheral axotomy: a new clinical approach in the management of peripheral nerve trauma.
Electron microscopy of axotomised DRGs demonstrated that ALCAR treatment improves neuronal morphology, and in particular that as the dose increases, the morphological features of cell death are obviated.
Such preservation of mitochondrial morphology suggests that ALCAR does indeed block the cell death pathway at the mitochondrial level. This is likely by limiting bioenergetic dysregulation and hence the production of reactive oxygen species, sufficiently to prevent mitochondrial death, and the resultant release of apoptosis inducing factors that are thought to be the final trigger to neuronal death via caspase activation. The presence of some intact, swollen mitochondria and a degree of ER dissolution in even 50 mg/kg/day ALCAR treated groups merely reflects the normal regenerative response to axotomy, preparing the cell for altered protein synthesis and increased metabolic activity.
Axotomy caused a reduction in the neuron-containing volume of L4+L5 DRGs. The extent of volume loss varied with dose of ALCAR, with results reflecting those obtained from neuronal cell counts since DRG volume normalised as dose of ALCAR increased. DRG volumes at doses above 5 mg/kg/day were not significantly different compared to the contralateral, non-axotomised controls at the 2 week time point examined. The volume loss with the lowest dose (0.5 mg/kg/day), and with sham treatment, are in accordance with published results after no treatment.
ALCAR administration is postulated to prevent the loss of DRG volume that results from axotomy by preserving neuronal numbers and perhaps also by maintaining the volume of the surviving neurons which would otherwise decrease after axotomy.
The spatial relationship of the DRG population is therefore preserved, facilitating continuation of normal intercellular interactions between neurons and glial cells. This may avoid imbalances in the concentration of local trophic factors, optimising the environment for regeneration.
The precise mechanism of action by which ALCAR prevents sensory neuronal loss is not yet known. Some suggest a role involving increased neurotrophin responsiveness, hence reducing the effect of losing distal neurotrophic support which occurs after axotomy.
Glucose and leucine uptake in the hypoglossal nucleus after hypoglossal nerve transection with and without prevented regeneration in the Sprague-Dawley rat.
Memory loss in old rats is associated with brain mitochondrial decay and RNA/DNA oxidation: partial reversal by feeding acetyl-l-carnitine and/or R-alpha-lipoic acid.
Memory loss in old rats is associated with brain mitochondrial decay and RNA/DNA oxidation: partial reversal by feeding acetyl-l-carnitine and/or R-alpha-lipoic acid.
further enhancing its clinical application to even include peripheral nerve trauma presenting after completion of the period of neuronal death.
In the clinical setting, ALCAR has been shown to be a safe and effective pharmacotherapeutic agent when administered orally, or parenterally. It improves nerve conduction velocities in both motor and sensory neuropathies
Immunohistochemical quantification of cutaneous innervation in HIV-associated peripheral neuropathy: a study of acetyl-l-carnitine therapy. Abstract 36. Third International Workshop on Salvage for HIV Infection, Chicago 2000.
Immunohistochemical quantification of cutaneous innervation in HIV-associated peripheral neuropathy: a study of acetyl-l-carnitine therapy. Abstract 36. Third International Workshop on Salvage for HIV Infection, Chicago 2000.
In conclusion, ALCAR eliminates neuronal loss in a dose-dependent fashion after neurometsis, acting via a mechanism involving mitochondrial protection. This strong evidence of a dose-response supports earlier findings that ALCAR is neuroprotective
Systemic acetyl-l-carnitine eliminates sensory neuronal loss after peripheral axotomy: a new clinical approach in the management of peripheral nerve trauma.
and may prove useful in choosing a dose for clinical use. Combined with its promotional effect on nerve regeneration and its clinical safety profile, ALCAR represents a clinically applicable pharmacological agent for potential use in improving the outcome of peripheral nerve injuries.
Acknowledgements
This work was funded by the Swedish Medical Research Council (grant 02286), County of Vasterbotten, Ane'rs Foundation, and the East Grinstead Medical Research Trust (Registered Charity No. 258154). The authors wish to thank Dr Lev N. Novikov and Mrs Gunnel Folkesson from the Department of Integrative Medical Biology, Umeå University, Sweden for the electron microscopy contribution to this study.
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Abstr Eur J Plast Surg.2003; (In Process Citation)
Systemic acetyl-l-carnitine eliminates sensory neuronal loss after peripheral axotomy: a new clinical approach in the management of peripheral nerve trauma.
Immunohistochemical quantification of cutaneous innervation in HIV-associated peripheral neuropathy: a study of acetyl-l-carnitine therapy. Abstract 36. Third International Workshop on Salvage for HIV Infection, Chicago 2000.
Acetyl-l-carnitine deficiency as a cause of altered nerve myo-inositol content, Na, K-ATPase activity, and motor conduction velocity in the streptozotocin-diabetic rat.
Glucose and leucine uptake in the hypoglossal nucleus after hypoglossal nerve transection with and without prevented regeneration in the Sprague-Dawley rat.
Memory loss in old rats is associated with brain mitochondrial decay and RNA/DNA oxidation: partial reversal by feeding acetyl-l-carnitine and/or R-alpha-lipoic acid.
☆Dose-response relationship of acetyl-l-carnitine and sensory neuron rescue after peripheral nerve injury. Mr A.D.H. Wilson, Mr A. Hart, Prof. M. Wiberg, Dr G. Terenghi (London/Umeå), British Association of Plastic Surgeons, Winter meeting, 27 November 2002.
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